Increasing evidence on long-term health outcomes following SARS CoV-2 infection shows post-viral symptoms can persist for months. These symptoms are often consistent with those of Myalgic Encephalomyelitis or Chronic Fatigue Syndrome (ME/CFS). The aim of the present study was to examine the prevalence and outcome predictors of post-viral fatigue and related symptoms 3- and 6-months following symptom onset.n = 88) were recruited from a Post-COVID-19 Respiratory Clinic (PCRC) in Vancouver, Canada to examine predictors of long-term fatigue and substantial fatigue. Multivariable mixed effects analyses examined the relationship between patient predictors, including pre-existing comorbidities, patient reported outcome measures, and fatigue and substantial fatigue at follow-up.A prospective cohort of patients hospitalized with Coronavirus disease (COVID-19)  and 14 (16%) respectively. At 6 months these numbers declined to 47 (60%) patients experiencing fatigue and 6 (6%) experiencing substantial fatigue. Adjusted analysis, for sex, age, and time, revealed the number of pre-existing comorbidities to be associated with fatigue  and substantial fatigue  at 3 months follow-up. Except for shortness of breath, self-care, and follow-up time, all follow-up variables were found to be associated with fatigue and substantial fatigue at 3 months.Fatigue and substantial fatigue are common after COVID-19 infection but often diminish over time. A significant number of patients continue to exhibit long-term fatigue at 6 months follow-up. Further research is needed to clarify the causality of viral infections in the development and severity of fatigue as a symptom and in meeting post-viral fatigue syndrome or ME/CFS diagnostic criteria. Participants included those with a confirmed SARS-CoV-2 infection who were hospitalized from March to June 2020. Detailed methods involving this cohort as well as 3- and 6-month respiratory outcomes, and some patient-reported outcome measures (PROMs) have been previously reported elsewhere , 14. BriThe EQ-5D is a five dimension preference-based and quality-of-life assessment. Scores from the EQ-5D were converted to a health utility index whereby scores of 1 represent perfect health and scoring of 0 is death . The UCS2.2.Primary outcomes included the presentation and severity of persistent fatigue at 3- and 6-months post-symptom onset. Fatigue was classified as an individual reporting feeling tired or having little energy for several days or more over the last 2 weeks (PHQ-9) as well as, indicating “always feeling tired” (FI). Substantial fatigue was classified as an individual reporting fatigue, as defined above, and “slight” differences in their ability to conduct usual activities (EQ-5D).2.3.n = 6), modeling for substantial fatigue included only the 3-month follow-up period. McNemar’s test was then used to examine changes in the presence of substantial fatigue across time. Relationships between clinical predictors and PROMs were examined using multivariable mixed effects linear regression modeling. Statistical significance was determined by a two-sided p-value of <0.05. All analyses were conducted using the statistical software package R (Version 4.2.1).Descriptive statistics were used to describe participant characteristics at the time of hospitalization. Associations between all predictors  and presentation of fatigue and substantial fatigue at 3- and 6-months were examined using multivariable mixed effect logistic regression modeling to account for correlations between timepoints and bivariate analyses were used to determine potential discrepancies between those with and without fatigue at each timepoint. Adjusted analysis of all predictor variables at 3- and 6-month timepoints did not include the EQ-5Ds “self-care” dimension due to a lack of variance in responses. Given the low number of patients with substantial fatigue at 6 months follow-up (3.n = 88), mean age was 61.1 (±16.2) years, 63.6% were male, 45.5% identified as white, 80.7 and 89.8% had no pre-existing lung or autoimmune disease, respectively. Approximately 48.2% were admitted to ICU, of whom 20.5% were on mechanical ventilation during hospitalization. Prevalence of fatigue and substantial fatigue were reported to be 66.7% and 16.1%, respectively, at 3 months. By 6-month follow-up, fatigue was exhibited in 59.5% and substantial fatigue in 6.9% of patients. Among participants with and without fatigue at 3- and 6- months, bivariate analysis indicated statistically significant differences in participant reported scoring on PHQ-9, EQ-5D, shortness of breath, and overall health scoring ( scoring .3.1.p = 0.06). Adjusted multivariable analysis, controlling for age, sex, and time at the point of hospitalization, revealed number of comorbidities  to be a predictor of fatigue at 3 months post-viral infection (p = 0.012). Patients who did not exhibit fatigue at 3 months did not go on to exhibit fatigue at 6 months follow-up.The number of pre-existing comorbidities had a trend toward association with fatigue at 3 months (nfection . Interes3.2.Adjusted analysis, controlling for age and number of comorbidities, revealed correlations between fatigue and all variables measured at 3- and 6-months, with the exception of self-care and follow-up time .3.3.p = 0.033, Fourteen individuals exhibited substantial fatigue at 3 months and six continued to exhibit substantial fatigue at 6 months follow-up. No patients developed new substantial fatigue between follow-up time periods. Adjusting for age, the number of pre-existing comorbidities at hospitalization was associated with substantial fatigue at 3 months follow-up (OR 1.73; 95% CI 1.06–2.95; 3.4.With the exception of shortness of breath, all follow-up variables were associated with substantial fatigue at 3 months. Due to the small number of cases at 6 months, we did not examine this relationship further.4.The present study shows that long-term fatigue can persist for at least 6 months in 59.5% of patients previously hospitalized with SARS-CoV-2 infection. Within the PCRC cohort, reductions in the prevalence of fatigue and substantial fatigue were observed between 3- and 6-months follow-up. Greater improvements were observed among those experiencing substantial fatigue, which decreased to less than half of the original prevalence by the 6-month follow-up timepoint. These findings align with existing literature reporting a decreasing trend in the proportion of individuals experiencing fatigue across weeks since acute presentation , 21. PatWhile contrary to some findings, an unexpected and subtle protective effect of age on the presence of fatigue was observed at follow-up timepoints. This finding is consistent with Subramanian et al.  who founMultimorbidity has also previously shown associations with post-viral fatigue syndrome symptoms , 22. Our4.1.Our findings highlight the importance of further examinations into the role viral infections have in the presentation of long-term fatigue and related multimorbidities. While the number of individuals experiencing fatigue after viral infection is expected to decrease at follow-up timepoints, there are a subset of individuals for whom fatigue presentation and severity will persist. It is important that clinical teams remain attentive to monitoring patients for long-term fatigue after infection with SARS-CoV-2, encouraging patients to engage in practices that can aid in mitigating fatigue severity and lasting post-COVID symptoms. The considerable proportion of patients within our study continuing to exhibit symptoms of fatigue at 6 months highlights the need for further investigations into evidence-based practices that can meet the needs of those experiencing long-term fatigue after acute viral infection. Despite the change in severity with newer variants of SARS-CoV-2, it is key for provincial initiatives such as the Post-COVID-19 Interdisciplinary Clinical Care Network and national networks to support continuous data collection to assist longitudinal studies in Long COVID. Our findings add to the growing body of literature illuminating the role viral infections, such as COVID-19, may have in the development of long-term symptoms and persisting fatigue.4.2.This study has several limitations. Namely, our findings may be limited by the small participant sample size. As such, we were unable to examine correlates of substantial fatigue at 6 months due to limited size and subsequent lack of power. This prohibited examination of differences in the predictors of substantial fatigue at 3- and 6-months follow-up and prevented further investigation into those no longer exhibiting substantial fatigue at 6 months. Likewise, given our inability to ascertain whether criteria for ME/CFS or post-viral fatigue were met, substantial fatigue was used as a proxy. It is therefore likely that the prevalence of post-viral fatigue syndrome found in this study is substantially higher than rates adhering to strict clinical guidelines. Gaps remain in knowledge surrounding which comorbidities may predispose individuals to greater levels of long-term fatigue and its severity after hospitalization. Several studies have identified associations between self-reported measures and fatigue related outcomes, including reductions in quality of life and cognitive impairment . However5.After hospitalization, a significant proportion of individuals recovering from COVID-19 will continue to experience lingering symptoms for up to 6 months. For many patients, the presentation of post-viral symptoms will manifest in increased levels of fatigue. Further investigation into the presentation of fatigue, post-COVID infection, is needed to support evidence-based care and management for individuals experiencing long-term post-viral symptoms.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the University of British Columbia Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.AA performed the statistical analysis. TM and LN wrote the first draft of the manuscript. EM wrote a section of the manuscript. TB organized the database. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lavandula austroapennina N.G. Passal., Tundis and Upson has aroused interest. An insight into the chemical composition of the corolla, calyx, leaf, stem, and root organs was carried out following ultrasound-assisted maceration in n-hexane. The obtained lipophilic extracts were explored using ultra-high-performance chromatography coupled to high-resolution mass spectrometry (UHPLC-ESI-QqTOF-MS/MS). The extracts from the different organs varied in their relative content of fatty acids, ursanes, and oleanane-type triterpenes. In particular, the oleanolic acid content appeared to increase in the order of corolla < leaf < stem. An MTT assay was performed to verify the possible cytotoxicity of the organ extracts of L. austroapennina at a concentration ranging from 12.5 to 400 µg/mL on the Vero CCL-81 cell line. Antiviral activity against herpes simplex virus type 1 (HSV-1), alpha human coronavirus 229E (HCoV-229E), and poliovirus type 1 (PV-1) was evaluated via a plaque reduction assay in the same cellular model. All the extracts did not show cytotoxic effects after 2 and 24 h exposure times, and the antiviral efficacy was particularly important for the stem extract, capable of completely inhibiting the tested viruses at low doses. The antiviral activity in a non-enveloped virus PV-1 allowed the assertion that the extracts from the organs of L. austroapennina, and especially the stem extract, interfered directly with the viral envelope. This study underlines how much knowledge of a territory’s medicinal plant heritage is a harbinger of promising discoveries in the health field.In a framework aimed at the recovery and enhancement of medicinal plants endemic to the territory of the Cilento and Vallo di Diano National Park,  Emerging and re-emerging viral infections remain a large-scale challenge all over the world. The emergence arises from rapidly evolving pathogens and changes in the environment, as well as in a host providing such agents with suitable new ecological niches. The last decades have recorded unprecedented pandemic outbreaks: severe acute respiratory syndrome (SARS) in 2003, H1N1 “swine” influenza (2009), Middle East respiratory syndrome (MERS) in 2012, chikungunya (2014), and Zika (2015), as well as the last pandemic explosion due to SARS-CoV-2 (2020 to the present). Alongside these infections, many past emerging infectious agents stably co-exist with humans, and it is demonstrated by the persistence of endogenous retroviruses in human DNA -2,5 diphenyl tetrazolium bromide (MTT) assay  based on the manufacturer’s instruction. Basically, 2 × 104 cells/well were seeded in 96-well plates and incubated overnight (O/N) at 37 °C in a humidified atmosphere. Cells were treated with different concentrations  of each extract for 2 and 24 h. Then, cell monolayers were treated with MTT solution (0.5 mg/mL) for 3 h in the dark. The amount of soluble formazan was evaluated by recording the absorbance (Abs) at the wavelength of 570 nm with a TECAN M-200 reader . The cell survival was calculated by applying the following formula:African green monkey kidney epithelial cell line (Vero CCL-81) was purchased from American Type Culture Collection . Vero cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L of glucose  supplemented with 10% heat-inactivated fetal bovine serum , 2 mM of L-glutamine (Microtech), and 100 IU/mL of penicillin–streptomycin solution  and maintained at 37 °C in a humidified atmosphere with 5% COHSV-1 strain SC16, HCoV-229E (ATCC VR-740), and poliovirus type 1 (PV-1) strain Chat (ATCC VR-1562) were propagated on Vero CCL-81 cells.5 cells/well) were seeded in 24-well plates and incubated O/N. The monolayer was treated with different dilutions of the compounds, ranging from 200 to 0.8 µg/mL, and infected with viruses at 0.01 multiplicity of infection (MOI). Following a 1 h adsorption at 37 °C, the monolayer was washed with acidic citrate buffer (pH 3.0) to inactivate non-penetrated viruses and incubated for 24–48 h at 37 °C in DMEM supplemented with 3% carboxymethylcellulose (CMC). Finally, cells were fixed with 4% formaldehyde and stained with 0.5% crystal violet for the detection of plaques. A set of treatments  calculated with GraphPad Prism (version 5). Statistical differences were evaluated via one-way ANOVA followed with a Dunnett test; a value of n-hexane extracts from the corolla, calyx, stem, leaf, and root of L. austroapennina. TOF-MS and TOF-MS/MS data of tentatively identified compounds are reported in UHPLC-ESI-Qq-TOF-MS/MS analysis was carried out to unravel the chemical composition of the 1, 2, 5, and 7 were likely rosmaridiphenol, rosmanol, rosmaquinone B, and carnosic acid, respectively  with the deprotonated molecular ion at m/z 255.2334 was tentatively identified, beyond the monounsaturated oleic acid (19) with [M-H]− at m/z 281.2489. These compounds, together with compounds 13 ([M-H]− ion at m/z 277.2178) and 16 ([M-H]− ion at m/z 279.2334)—likely linolenic and linoleic acid, respectively—were previously found as constituents of apolar extracts of the stem and leaf of L. officinalis 14 and 15 (m/z 455.3539(6), were identified as oleanolic acid and ursolic acid, respectively, based on the comparison of their relative retention time with those of pure reference compounds. These compounds were abundant in all the investigated organs and, comparably to a previous observation of L. pubescens, oleanolic acid appeared to be highly present in the stems and leaves —the fragment ion at m/z 409.3113, likely due to the loss of vicinal diol function in A-ring and water from the deprotonated molecular ion [9 and 10 were tentatively identified as dioxo-hydroxyurs-12-ene-28-oic acid and 3-oxo-hydroxyurs-12-en-28-oic acid, respectively  was the most abundant fatty acid in the calyx fraction, whereas its content decreased in the leaf extract and, more markedly, in the stem and root fractions. Corolla and calyx extracts also contained a comparable amount of linoleic acid (16), while corolla was distinguishable for its content in linolenic acid (13) and the appreciable oleic acid (19) abundance. Similarly, a differentiation of the triterpene constituents showed that ursolic acid (15) was highly contained in the corolla, and calyx decreased in leaf extract, and more markedly, in stem and root mixtures, whereas its constitutional isomer—oleanolic acid (14)—increases relatively from the calyx to the leaf, reaching the highest percentage content in stem.The relative quantization, which was calculated taking into account the sum of the individual metabolites by metabolic class identified, highlights in particular that the calyx extract differed in the higher content of triterpenes . A metabL. austroapennina organs cytotoxicity through an MTT test. Vero cells were used due to their susceptibility to several types of viruses as cell substrates for human vaccines [L. austroapennina organ extract affected cell viability significantly at both time points. Only the corolla, at 400 and 200 µg/mL, exhibited a slight toxicity after 24 h of incubation.Vero cell lines were preliminarily used for carrying out the in vitro evaluation of vaccines  due to tvaccines . An MTT L. austroapennina apolar extracts, two types of viruses that were mainly responsible for human infections, namely HSV-1 and HCoV-229E, were used as models for enveloped DNA and RNA viruses, respectively.In order to explore the antiviral potential and elucidate the most likely mechanism of action of As shown in Studies performed on HCoV-229E displayed that the extracts showed a general trend comparable with HSV-1 results. In detail, as reported in 90 equal to 30.3 µg/mL.Surprisingly, in the pre-treatment test, even fewer active extracts showed a great viral inhibition. The stem extract displayed the best activity among the others, with an ICThese promising preliminary results suggested a possible involvement of the extracts in an early and extracellular stage of the viral life cycle before the virus penetration. Then, we performed a cell pre-treatment and post-treatment assay against the same viruses. The results are shown in L. austroapennina extracts act directly on viral particles before entry in the host cell. Since the two selected viruses (HSV-1 and HCoV-229E) are both enveloped viral models, the extracts’ mode of actions were further investigated by performing a plaque reduction assay in the best condition of activity (pre-treatment) with a non-enveloped virus, namely PV-1.The data confirm that The graphs in L. austroapennina organs exhibited antiviral activity on both HSV-1 and HCoV-229E. The former is a double-stranded DNA, enveloped virus, responsible for labial herpes, and rarely constitutes the causative agent of more severe diseases, such as keratitis and encephalitis which are often fatal in immunocompromised people [L. austroapennina organs is in line with the data by Yucharoen et al. [L. officinalis (10%), Hypericum perforatum, Glycyrrhiza glabra, Melissa officinalis, Eleutherococcus senticosus, and Sarracenia (mixed species) was able to reduce the oro-facial herpes symptoms (72 h) and the frequency of future outbreaks [The apolar extracts from d people . HSV infd people . The avad people . Thus, tn et al. , who revutbreaks .L. austroapennina extracts. This enveloped virus has a single-stranded RNA genome and causes epidemic outbreaks during winters [L. austroapennina apolar extracts, it was observed that the highest concentration of both corolla and calyx extracts exhibited a significant viral replicative inhibition, while stem and leaf extracts were active at 100 and 50 µg/mL, respectively, and the root sample exerted virus inhibition close to 50% at 12.5 µg/mL. The extracts appeared to be more effective when a pre-treatment experiment was carried out, with the only exception of the root sample, which showed a similar trend. The root extract appeared peculiarly enriched in docosyl ferulate (20). This compound was shown as a behaviourally active GABAA receptor complex (GABAAR) agonist [Lavandula species.HCoV-229E infection has been also affected by  winters . It belo winters . The las winters . When a  agonist , whose oLavandula species against HCoV-229E, there are various reports in relation to SARS-CoV-2 and avian influenza virus (H5N1), which share similar morphology (RNA-virus with envelope) and symptoms (respiratory disease). In this regard, essential oils are of interest as phytochemicals that are able to interfere with the enveloped viral lipid bilayer or inhibit specific processes in the replication cycle, preventing viral cellular shedding [Lavandula angustifolia and Salvia officinalis were found to be active against H5N1 [L. angustifolia, L. stoechas, and L. × heterophylla EOs—were shown to inhibit ACE-2 and LOX enzymes involved in the SARS-CoV-2 infection [Lavandula latifolia reduce the symptoms of acute respiratory infections by viruses belonging to the Coronaviridae family. In fact, the treatment of patients with Tavipec® capsules, providing 900 mg of EO from the flowering tops and stems of spike lavender, allowed the observation of an improvement in the symptoms of acute rhinosinusitis and a decrease in the symptoms of acute bronchitis versus a placebo [Stem sample at 12.5 and 25 µg/mL induced viral inhibition of ~60% and ~90%, respectively. Although there are no data on the antiviral activity of the shedding . Recentlnst H5N1 , and linnfection . Clinica placebo .L. austroapennina organs differently induce viral suppression acting in the early stages of a viral replication, through disruption of the pathogen envelope, which is derived from the host cell plasma membrane. In fact, fatty acids, which represent the most abundant component for almost all the extracts investigated, were capable of inducing the inactivation of some enveloped viruses [L. austroapennina. The effect of triterpenes cannot be excluded. Ursolic acid and oleanolic acid have been shown to prevent virus adsorption and invasion into host cells in the early stages and to inhibit the viral replication process after cell infection [The antiviral drugs can exploit different mechanisms such as the inhibition of virus attachment, entry and uncoating, or it can interfere with viral enzymes, such as polymerase and protease as well as reverse transcriptase and integrase . Data he viruses ,52. Medi viruses . Fatty a viruses . The mos viruses . Palmiti viruses , and frenfection . Oleanolnfection ,59. Bothnfection . Ursolicnfection . The effnfection .90 value equal to 30.3 µg/mL. This study underlines how much knowledge of a territory’s medicinal plant heritage is a harbinger of promising discoveries in the health field. On the other hand, the definition of the chemical profile of a plant extract is mandatory to obtain an optimal recovery and exploitation of the bioactive components.This study underlines how much knowledge of a territory’s medicinal plant heritage is a harbinger of promising discoveries in the health field. On the other hand, the definition of the chemical profile of a plant extract is mandatory to obtain an optimal recovery and exploitation of the bioactive components. The chromatographic analyses in high-resolution mass spectrometry showed that the extracts were discriminable on the basis of the relative amount of the identified fatty acids and triterpenes. In particular, oleanolic acid was the most abundant triterpene in the stem extract, and its content was higher than that of ursolic acid in the leaf, whereas a comparable content of ursolic acid and oleanolic acid was in the corolla. The calyx extract was distinguished by the palmitic acid content. The extracts were evaluated for cytotoxicity on the Vero cell line and analyzed for their antiviral capacity against HSV-1 and HCoV-229E infection. The stem extract showed an interesting antiviral efficacy in the pre-treatment assay, and its dose equal to 3.1 µg/mL completely inhibited HSV-1 replication. Analogously, against HCoV-229E, the stem extract displayed a strong activity exhibiting an IC


Photodynamic therapy is a non-invasive therapeutic strategy that combines external light with a photosensitizer (PS) to destroy abnormal cells. Despite the great progress in the development of new photosensitizers with improved efficacy, the PS’s photosensitivity, high hydrophobicity, and tumor target avidity still represent the main challenges. Herein, newly synthesized brominated squaraine, exhibiting intense absorption in the red/near-infrared region, has been successfully incorporated into Quatsome (QS) nanovesicles at different loadings. The formulations under study have been characterized and interrogated in vitro for cytotoxicity, cellular uptake, and PDT efficiency in a breast cancer cell line. The nanoencapsulation of brominated squaraine into QS overcomes the non-water solubility limitation of the brominated squaraine without compromising its ability to generate ROS rapidly. In addition, PDT effectiveness is maximized due to the highly localized PS loadings in the QS. This strategy allows using a therapeutic squaraine concentration that is 100 times lower than the concentration of free squaraine usually employed in PDT. Taken together, our results reveal the benefits of the incorporation of brominated squaraine into QS to optimize their photoactive properties and support their applicability as photosensitizer agents for PDT. All the samples were diluted in ethanol to disrupt the membrane and release all the entrapped dye molecules. The concentration C of Br-Sq-C12 was determined using the Lambert–Beer law (A = ε l C), knowing that the extinction coefficient (ε) of Br-Sq-C12 in EtOH is 290.484 M−1 cm−1.The concentration of dye entrapped in QS was determined by measuring the UV–Vis absorbance The dye-loading coefficient was determined by lyophilization of the samples  at 193 K and 5 Pa for 5 days. Then, the samples were weighted, and the loading in mass was determined through the following equation.Br-Sq-C12, different dilutions in ethanol were prepared from a stock solution (0.5 mM). The absorbances were measured and their maxima were plotted vs. the sample concentration, being the slope of the linear fitting of the molar extinction coefficient (ε). The analysis was performed in duplicate and data were considered acceptable when the difference between the measured log ε was equal to or lower than 0.02 to their average.To determine the molar extinction coefficient of the Br-Sq-C12 and dye-loaded QSs in acetone, absolute ethanol (EtOH), methanol (MeOH), double distilled water (ddH2O), and Dimethyl sulfoxide (DMSO). The absorption was measured at room temperature by UV–Vis spectroscopy  in the range of 500–800 nm using quartz cuvettes, using a 1 cm path length.The determination of the solvatochromism was performed by preparing different solutions of Br-Sq-C12 and dye-loaded QS. The excitation wavelength was different depending on the solvents and was set at the squaraine hypochromic shoulder previously recorded at the UV–Vis spectra. The excitation and emission slits were 5 nm and 5 nm, respectively.Fluorescence emission measurements were acquired in steady-state mode and recorded in the range of 595–750 nm using a Horiba Jobin Yvon Fluorolog 3 TCSPC fluorimeter  equipped with a 450-W Xenon lamp and a Hamamatsu R928 photomultiplier  by using solvents with different polarity to investigate the solvatochromic behavior of both the Br-Sq-C12 and ddH2O for the dye-loaded QSs. The analyzed samples had an absorbance of around 0.1 to avoid aggregations/fluorescence quenching. The final result is an average of three independent measurements of different dye solutions.Fluorescence quantum yields (QY) were determined using the same instrument with Quanta-φ integrating sphere and De Mello method. The QY was evaluated in absolute ethanol for the Br-Sq-C12 and ddH2O for the dye-loaded QSs.Fluorescence lifetimes (LT) were determined using the time-correlated single photon counting method  using a 636 nm Horiba Jobin Yvon NanoLED  as the excitation source and a pulse repetition frequency of 1 MHz positioned at 90° with respect to a TBX-04 detector. Lifetimes were calculated using DAS6 decay analysis software. The LT was evaluated in absolute ethanol for the Br-Sq-C12  were determined by DLS, while the determination of the ζ-potential values (z-pot) was performed with the ELS. Both measurements were carried out using a Zetasizer Ultra . The measurements with the DLS technique were performed using a fluorescence filter to block the light resulting from fluorescence emission, which may alter the correlation function (the instrument exploits a 633 nm laser). Meanwhile, the ELS measurements were performed employing A DTS1070 folded capillary cell  was used, applying a voltage of 40 mV between the gold electrodes, and being calculated using the Helmholtz–Smoluchowski equation, which can potentially underestimate the real zeta-potential [The mean size and size distribution of the QS loaded with 200 and 300 μM otential ,53. All Cryogenic transmission electron microscopy (cryo-TEM) images were acquired with a JEOL JEM microscope  operating at 200 kV under low-dose conditions. First, 10µL of the sample was deposited onto the holey carbon grid and, immediately after, vitrified by rapid immersion in liquid ethane. The vitrified sample was mounted on a cryo-transfer system and introduced into the microscope . Images were recorded on a CCD camera .1O2 forming the colorless o-dibenzoylbenzene derivative. The 1O2 scavenger activity can be monitored through a decrease in the electronic absorption band of DBPF at 415 nm. Stock solutions were prepared in DMSO, absolute ethanol, and phosphate buffer , respectively, for DPBF, free Br-Sq-C12, and Br-Sq-C12-loaded quatsomes. Each solution was then diluted in phosphate buffer  to obtain a DPBF concentration of 25 μM and a final concentration of 2.5 μM for both the free and the encapsulated dye. The solutions were placed in a 1 cm quartz cell and irradiated at various time intervals under stirring in an aerated solarbox . The light was filtered in an optical filter with a 515 nm cut-off to avoid DPBF degradation. At predefined time points , absorption spectra were recorded on a Cary 300 Bio spectrophotometer instrument . The decrease in the DBPF absorption contribution at 415 nm was plotted as a function of the irradiation time.As a probe molecule, 1,3-Diphenylisobenzofuran  was used to evaluate Reactive Oxygen Species (ROS) generation by following the protocol previously described in the literature . DPBF ra2 at 37 °C, using Falcon™ plates as supports.Human breast adenocarcinoma cell line  was cultured in DMEM High Glucose  growth medium complemented with 10% Fetal Bovine Serum , 100 mg/mL PenStrep , and 2 mM L-glutamine . Cells were cultured in a humidified incubator  with 5% CO4 cells/well) were seeded in 96-well plates . Six hours after plating, cells were treated with QS_Blank at two different membrane components’ (Chol + Stk) final concentrations (10 μg/mL and 2 μg/mL). Cell viability was assessed using CellTiter 96® Aqueous Non-Radioactive cell proliferation assay  according to the manufacturer’s instructions 24, 48, and 72 h after treatment. Briefly, 2 h after MTS incubation at 37 °C, absorbance at 490 nm was recorded using a microplate reader . Absorbance values were normalized on the control at 24 h and analyzed as being proportional to the number of viable cells. Similarly, the cytotoxicity of QS (2 μg/mL) loaded with increasing dye concentrations  were seeded in 96-well plates. Six hours after plating, cells were treated with QS_Sq at the concentrations reported in Br-Sq-C12 in its free form at the same concentrations. After O/N incubation at 37 °C and 5% CO2, the cells were irradiated for 15 min with a RED-LED array  specifically designed and produced by Cicci Research s.r.l . Cell viability was assessed 24, 48, and 72 h after irradiation using CellTiter 96® AQueous Non-Radioactive cell proliferation assay  as described above. The photodynamic effect of Br-Sq-C12-loaded QS was evaluated by comparing the viability of cells treated with QS_Sq or with the same concentration of Br-Sq-C12 in its free form upon irradiation. For each condition, eight technical replicates were set up and three independent experiments were performed.To evaluate the photodynamic effect of QS_Sq, MCF-7 cells  has been used to label and track the whole cellular volume in MCF-7 live cells. Briefly, MCF-7 cells were seeded in Ibidi μ-Slide 8 wells (1.6 × 104 cells/well), and 24 h after seeding, cells were treated overnight with 85 nM of Br-Sq-C12 in its free form or incorporated within 2 μg/mL QS (QS_Sq_200). After the incubation with the QS_Sq_200, the cells were washed twice with PBS and incubated with Calcein (500 nM) for 30 min, washed twice with Hanks’ Balanced Salt Solution (HBSS), and fixed in 4% paraformaldehyde (PAF) at 37 °C for 2 min. The cells were observed using a Leica TCS SP8 confocal system  equipped with an HCX PL APO 63X/1.4 NA oil-immersion objective. To simultaneously detect the probes, Br-Sq-C12 was excited with a HeNe laser at 633 nm, whereas Calcein was excited with a DPSS laser at 561 nm. Images were acquired on the three coordinates of the space (XYZ planes) with a resolution of 0.081 μm × 0.081 μm × 0.299 μm and were processed and analyzed with ImageJ software . Three-dimensional images with Calcein allowed for assessing whether the Br-Sq-C12 encapsulated into QS or in its free form was included within the cellular volume or not.To verify the intracellular uptake of Sq-loaded QS and compare it with that of the dye in its free form, Calcein . Differences with p-values < 0.05 were considered statistically significant and *: p-value < 0.05, ***: p-value < 0.0005, ****: p-value < 0001.Data are shown as the average values of three independent pulled experiments ± standard error mean (SEM). Statistical analyses were performed using Graph-Pad Prism 6.0 software . The statistical significance between different conditions was determined by performing a Br-Sq-C12, was developed. By the incorporation of the longer alkyl chain, higher hydrophobicity is provided, hypothesizing that this would promote its stable incorporation in the vesicular membranes. This study evidences the importance of the hydrocarbon chain length for the stabilization of the dye in a vesicular membrane, and in particular, into a quatsome nanovesicle.In a previous study, Bromo-Squaraine-C4 (Br-Sq-C4), a non-water soluble indolenine-based dye quaternarized with a four-carbon atom chain, demonstrated successful PDT results in vitro ,49. HoweBr-Sq-C12 dye (1), synthesized following a procedure reported in ref. [2. This reaction was performed under microwave irradiation and led to increased acidity of the methyl group promoting the following condensation reaction. The final dye was then obtained in a one-step reaction under microwave heating following our well-established method for indolenine-based squaraines [The synthesis of Bromo-Squaraine-C12, -C12 dye , started in ref. , to obtauaraines .Br-Sq-C12 were prepared using the DELOS-susp methodology [Br-Sq-C12; 200 and 300 μM as initial, pre-processing dye concentration. As a result, two batches of QS encapsulating Br-Sq-C12 were obtained, named based on their final post-processing dye concentration: QS_Sq_160 and QS_Sq_200, from the 200 and 300 μM, respectively  and Cholesterol (Chol), with a 1:1 molar ratio Stk/Chol, and loaded with hodology  at two dectively . In addiL), with an effective dye concentration of ~200 μM at the vesicles.All formulations showed a very similar concentration of membrane components . Sample n = 100). It is important to keep in mind that the averaged values obtained from cryo-TEM come from representative images, which help to confirm the data obtained from DLS being this last one more statistically representative.QS_Sq_160 and QS_Sq_200 were analyzed with DLS to determine the mean hydrodynamic diameter and polydispersity index (PDI). First, a systematic DLS study was performed in order to elucidate the optimal dilution for QS measurements. As detailed in the section “Systematic DLS study” included at the SI, DLS, and ELS measurements were performed at 1:10 dilution from the final formulation to ensure reliable results. Average hydrodynamic diameter (z-average), PDI, and ζ-potential average values are summarized in Br-Sq-C12 shows an absorption maximum at around 640 nm in ethanol (−1cm−1). The UV–Vis spectrum is characterized by a narrow absorption band in the NIR, an essential requirement for the PDT treatment, and a characteristic hypsochromic shoulder typical for polymethine dyes. The main absorption peak is associated with the π→π* HOMO–LUMO transitions, mainly localized on the squaraine core; on the other hand, the shoulder at higher energy can be ascribed to the HOMO-LUMO+1 transition [Br-Sq-C12 shows an excellent fluorescence emission with a maximum emission at 649 nm when dissolved in ethanol, although both the absorption and the fluorescence emission are completely quenched when dissolved in water due to an aggregation caused quenching (ACQ) effect. As shown in  ethanol a with a ansition ,60. As aansition , Br-Sq-CBr-Sq-C12, suggesting that the association with the QS did not change the energies and relative probabilities of the electronic transitions. The fluorescence quantum yield of Br-Sq-C12 in ethanol is in the ranges typical for squaraines in organic media. On the contrary, the QY in water cannot be detected due to the complete insolubility of the dye in aqueous media. However, after the entrapment in the QS, we were able to measure the QY in aqueous dispersion. The values are nevertheless very low, probably due to the presence of some H-aggregates, as also evidenced in the UV–Vis spectra reported in Br-Sq-C12 fluorescence lifetime showed a monoexponentially decay and is in the nanoseconds range, as already observed for several squaraines [The solvatochromic effect on both the dye and dye-loaded QSs absorption and emission spectra were also investigated and reported in uaraines .Br-Sq-C12 has been successfully incorporated into the QS membrane, allowing its fluorescence in an aqueous media.By comparing the values obtained for QS_Sq_160 and QS_Sq_200 in water, bi-functional equations were necessary to fit the decay curves, suggesting that two different types of interactions occurred with the QSs. Specifically, one fluorescence lifetime is slightly longer, while the other ones (ca. 85%) increased by ca. 2.7 times compared to the free dye. This longer decay could be ascribed to the decrease in rotational/twisting degrees of freedom, confirming a good degree of dye entrapment into the QS vesicles. On the other hand, the shorter lifetime could be due to the presence of a small amount of free dye on the QS surface, leading to a detrimental effect caused by the interaction with highly polar media, such as water . From thBr-Sq-C12-loaded QS with DLS and fluorescence spectroscopy, respectively. The obtained data proved the dye-loaded nanovesicular systems to be very stable for 18 months, with hydrodynamic diameter values around 90 nm and optimally low PDI values maintained around 0.2 , which can be indicative of the formation of dye aggregates. The results, presented in Similarly, photostability was evaluated with periodical UV–vis absorbance measurements for up to 18 months. We noticed a lowering of the main absorbance peak at 644 nm during the time for both samples in the study , indicatBr-Sq-C12 and dye-loaded QS to generate Reactive Oxygen Species (ROS) was carried out by using 1,3-diphenylisobenzofuran (DPBF) as a probe [Br-Sq-C12 is able to promote the complete decay of DPBF absorption within 180 s, while the same result was obtained in 10 min for reference RB. This fast ROS generation could be ascribed to the presence of bromine, which may facilitate the singlet to triplet state intersystem crossing due to the well-known heavy atom effect [Br-Sq-C12 into the QSs does not interfere with the ability of the dye to generate ROS rapidly, an essential requirement for PDT activity.A preliminary evaluation of the ability of both the free  a probe . DPBF ram effect . More imBr-Sq-C12-85nM-loaded quatsomes (QS_Sq_200) are slightly more cytotoxic than QS_Blank at 24 and 48 h after treatment with 2 μg/mL, although still highly biocompatible compared to QS_Blank at 10 μg/mL  was found to be the most active formulation, as it was significantly different from free Br-Sq-C12 at all monitored time points  was included within the cellular volume. As shown in Br-Sq-C12-loaded QS samples was not properly internalized . As a rA novel formulation based on Cholesterol–Sterealkonium QS loaded with a squaraine dye  has been proposed as an alternative photosensitizer in PDT. QS were prepared with green and scalable technology in a single step; the membrane components are available at pharmaceutical grade and readily adaptable for the incorporation of active targeting ligands. Considering the poor water solubility of squaraine dyes, their loading into QS offers an interesting strategy to bring them into aqueous media, enabling their use for bioapplications. Br-Sq-C12-loaded QS are stable for at least 18 weeks in aqueous media, showing modest dye leaking over time and weak photo instability. Moreover, we have demonstrated that Br-Sq-C12 incorporation into QS does not compromise its photophysical characteristics nor the ability of the dye to generate ROS rapidly, an essential requirement for PDT activity. Indeed, both the free Br-Sq-C12 and Br-Sq-C12-loaded QS possess faster and higher ROS generation ability compared to the well-known ROS generator Rose Bengal. Phototoxicity assays demonstrated that MCF-7 cells internalize Br-Sq-C12-loaded QS, and upon irradiation, an increase in the apoptotic/necrotic cell population is observed. The higher phototoxicity observed for the QS-loaded Br-Sq-C12 vs. its free form can be explained by the higher efficiency in PS delivery. QS provides more photostability in the aqueous media, higher cellular uptake, and significantly increases the local concentration of the PS. Indeed, the use of the nanocarrier system allows using much lower concentrations of photoactive dye (up to 10–1000 times lower). Taken together, our in vitro results support the applicability of QS as nanocarriers for PDT application and highlight the benefits of encapsulating squaraine dyes into stable nanostructures to optimize their photoactive properties.


Heterocephalus glaber) have unique physiological and behavioral characteristics that make them a species of interest in biomedical research. However, due to their small size, frequent blood sampling to monitor the animals is not possible. Thus, we aimed to validate enzyme immunoassays, tools which detect the quantity of hormone molecules, for monitoring stress-related hormones, glucocorticoids, and/or their metabolites, in naked mole-rats using urine and feces. We administered a high and low dose of adrenocorticotrophic hormone, which causes the animal to physiologically respond to stress, and a saline administration as a control, which should not elicit a physiological stress response. The results revealed two enzyme immunoassays, namely, a 5α-pregnane-3β,11β,21-triol-20-one detecting glucocorticoid metabolites with a 5α-3β-11β-diol structure and an 11-oxoaetiocholanolone detecting glucocorticoid metabolites with a 5β-3α-ol-11-one structure, for measuring stress-related hormones in male and female urine, respectively. Furthermore, one enzyme immunoassay, namely, 11-oxoaetiocholanolone detecting 11,17 dioxoandrostanes for measuring stress-related hormones in feces of both sexes. There were sex-related differences in how the animals responded to the high and low doses of the adrenocorticotrophic hormone. We recommend using feces to monitor stress-related hormones in naked mole-rats, which will be valuable when investigating housing conditions and other animal welfare aspects.Naked mole-rats  using urine and feces as hormone matrices. A saline control administration, and a high- and low-dose adrenocorticotropic hormone (ACTH) challenge were performed on six male and six female disperser morph NMRs. The results revealed that a 5α-pregnane-3β,11β,21-triol-20-one EIA detecting glucocorticoid metabolites (GCMs) with a 5α-3β-11β-diol structure is the most suitable assay for measuring concentrations in male urine samples, whereas an 11-oxoaetiocholanolone EIA detecting GCMs with a 5β-3α-ol-11-one structure appears the most suitable EIA for quantifying GCMs in female urine. An 11-oxoaetiocholanolone EIA detecting 11,17 dioxoandrostanes was the most suitable EIA for quantifying GCMs in the feces of both sexes. There were sex-related differences in response to the high- and low-dose ACTH challenge. We recommend using feces as a more suitable matrix for non-invasive GCM monitoring for NMRs which can be valuable when investigating housing conditions and other welfare aspects.Small size in mammals usually restricts long-term, frequent monitoring of endocrine function using plasma as a matrix. Thus, the non-invasive monitoring of hormone metabolite concentrations in excreta may provide an invaluable approach. The aim of the current study was to examine the suitability of enzyme immunoassays (EIAs) for monitoring responses to stressors in the naked mole-rat ( Heterocephalus glaber) is a monotypic genus in the subterranean African mole-rat family, the Bathyergidae, and is widely distributed across the horn of Africa including in Somalia, Kenya, Ethiopia, and Djibouti [The naked mole-rat  or their metabolites is useful as a tool to investigate the level of physiological stress and related welfare issues experienced by animals. Herein, stress is defined as a general syndrome occurring in response to any stimulus (stressors) that threatens or appears to threaten the homeostasis of an individual ,5. GlucoThere are multiple matrices that can be used for quantifying glucocorticoids or their metabolites such as plasma, feces, urine, hair/feathers, milk, saliva, and eggs ,14,15. FAlthough monitoring glucocorticoid metabolite concentrations non-invasively in matrixes such as urine and feces is a powerful tool, there can be dietary, species- and sex-specific differences in hormone metabolism and composition ,23. In aTwelve captive-bred naked mole-rats  housed at the Department of Zoology and Entomology, University of Pretoria, were involved in this study . These a® depot, Novartis, South Africa (Pty) Ltd.) solution were administered to the animals. There were two administration days; the first administration day (which was five days after the start of sample collection), all animals were intramuscularly injected with 200 µL of sterile physiological saline solution. Subsequently, all animals were randomly selected to receive either a low (20 ug/100 g body mass) or a high (60 ug/100 g body mass) dose of a synthetic ACTH solution 25 days later. The pre-calculated volume of synthetic ACTH was added to sterile physiological saline to result in a total of 200 µL for injection . At the beginning of the day, the animal was temporarily removed from the enclosure, and wood shavings were removed to avoid sample contamination and absorption. Thereafter, the enclosure was wiped down with 70% ethanol. The temporary handling was part of each animals’ cleaning routine and was performed as quickly as possible (≤5 min). At the end of each day, wood shavings were returned to the enclosures without removing the animal. On the two administration days (17th November and 11th December 2020), injections started at 10 am, and all produced urine and feces were collected up to 30 h post-injection. The animals were checked every 30 min for freshly voided urine samples which were collected using plastic pipettes labelled with individual animal ID. In addition, all freshly voided fecal samples (entire defecated sample) were collected using forceps. Individually labelled pipettes were rinsed with distilled water, and air-dried between samples whereas forceps were cleaned with ethanol (70%) and air-dried between each sample collection. Samples where feces and urine came into contact were discarded. Overall, 111 urine samples  and 87 urine samples  were collected during the ACTH administration and saline administration period, respectively. In addition, 217 fecal samples  and 204 fecal samples  were collected during the ACTH administration and saline administration period, respectively. The collected material was placed in individually labelled plastic vials, and frozen immediately upon collection at −20 °C.n = 30). In addition, samples from the same individual collected within 30 min of each other were combined (n = 17). Thereafter, suspensions were vortexed for 15 min and subsequently centrifuged at 1500× g for 10 min and the supernatant  formed was transferred into microcentrifuge tubes and stored at −20 °C for further analysis [Fecal samples were freeze-dried and subsequently pulverized using a mortar and pestle . Due to analysis .Defrosted urine samples and fecal steroid extracts collected during the ACTH administration period  were analyzed for immunoreactive uGCM and fGCM concentrations according to the procedure described by Ganswindt  using 5 Defrosted urine samples  were analyzed for immunoreactive uGCM using 5α-pregnane-3β,11β,21-triol-20-one and 11-oxoaetiocholanolone II EIA for males and females, respectively see . Similarn = 48). All urine steroid concentrations were expressed as mass per mg creatinine (Cr). All fecal steroid concentrations were expressed per mass of dry fecal matter.Creatinine concentrations in all native urine samples were determined by a modified Jaffe reaction ,38. SampThe EIA sensitivities and intra-assay and inter-assay coefficients of variation in high- and low-value quality controls for all EIAs are provided in To identify the suitable EIA for each matrix and sex, firstly, the baseline for each individual and each EIA was calculated. Following from the results of Edwards et al. , and due10 transformed prior to statistical analysis. In addition, the sexes were analyzed separately. A linear mixed-effects model, with ID as a random effect, was used to analyze if there was a significant change in uGCM or fGCM concentrations in relation to the interaction between time interval and dose using the lme4 package [p < 0.05. All statistical analyses were carried out using R statistical software with RStudio (2022.07.1) interface [Results from high or low ACTH administration and saline administration (collectively referred to as doses) for each sex were used to investigate the influence of dose-dependent responses. Firstly, individual uGCM and fGCM percentage change data were pooled using the median into baseline, 12-, 24-, 48-, 72-, 96-, and 120-h time intervals for each dose and sex. The time intervals were treated as categorical variables , corticosterone EIA (234 ± 128%), and 5α-pregnane-3β,11β,21-triol-20-one EIA (198 ± 48%), had an overall percentage increase above 100% for the males within a period of 48 h post-injection . On an iOf the five EIAs tested for fGCM quantification, the oxoaetiocholanolone I was identified as the most suitable EIA for both the male and female naked mole-rats, with the highest peak fGCM concentrations occurring within 48 h post-injection  of the variation in the percentage change in the uGCM concentrations and the interaction between the time and dose explained 43%  of the variation in the percentage change in the uGCM concentrations. The effect of the time period on the percentage change in the uGCM concentrations was dose-dependent  of the variation in the uGCM concentrations, and the interaction between the time and dose explained 28%  of the variation in the percentage change in the uGCM concentrations. The effect of the time period on the percentage change in the uGCM concentrations was not dose-dependent  of the variation in the fGCM concentrations, whereas the dose, time, and their interaction explained 27%  of the variation in the fGCM concentrations. The effect of the time periods on the percentage change in the fGCM concentrations was dose-dependent . The low-dose treatment had a peak at 12 h (207.36 ± 107.70) post-injection  of the variation, whereas the interaction between the dose and time explained 70% . The effect of the time periods on the percentage change in the fGCM concentrations was dose-dependent . The high-dose treatment revealed a peak at 24 h (160.54 ± 78.32) and 96 h (211.56 ± 33.07) post-injection , Palme et al. [Mus musculus f. domesticus), Touma et al. [Elephantulus myurus), Medger et al. [Cynomys ludovicianus), Crill et al. [To our knowledge, only five ACTH challenge test studies have so far investigated dose-dependent responses . Yet, ste et al.  did not a et al.  found thr et al.  found thr et al. . Interesl et al.  found thOur study found sex- and dose-dependent variations in the response to ACTH. In the analyzed male naked mole-rat urine samples, a peak in the GCM concentrations can be seen at 6 h post-injection in the animals who received either a low or high dose of ACTH. Urine GCM concentrations returned to the baseline after 24 h. Whereas in the male fecal samples, there was a peak at 12 h post-injection for the low dose, and a peak at 24 h for the high dose. Overall, the results here show the fGCM concentrations went below a 100% increase at 72 h and returned to the baseline after 96 h post-injection. The naked mole-rat male response to the doses is similar to the findings in cattle , wherebyCallithrix penicillate), results showed that females had an overall higher response to ACTH and saline administration compared to males [Rattus norvegicus), the 5α-pregnane-3β,11β,21-triol-20-one EIA was successfully validated for both sexes; however, high-performance liquid chromatography revealed that corticosterone metabolites differ between the sexes [Myodes glareolus), evidence suggests that the main excretory route of corticosterone is feces for males, whereas in females, excretion of corticosterone is evenly distributed between urine and feces [In all, it is not uncommon to see sex-specific differences in the non-invasive monitoring of stress-related hormones ,25,44. Fto males . Furtherto males . For exahe sexes . Finallynd feces . Overallnd feces  found nond feces . Conversnd feces , replicaAcomys cahirinus), laboratory mice, and meadow vole (Microtus pennsylvanicus), the peak in the fGCM concentrations occurred ~5 h [The determined peak GCM values post-administration for both sexes in both matrixes (6 h and 24 h) appear similar to the findings of Edwards et al. . The difred ~5 h , ~8 h [3red ~5 h  post-ACTred ~5 h  suggestired ~5 h ,52.The role of stress In cooperatively breeding species such as naked mole-rats is under much debate ,54,55. Oad libitum. Thus, these results could be a potential indication of high sodium concentrations [Unexpectedly, for both sexes, the results show there were some individuals for which there was no peak in the fGCM concentrations detected by the chosen 11-oxoaetiocholanolone EIA, but there was a peak in the uGCM concentrations and vice versa. A possible explanation for this observation could be changes in steroid metabolite excretion. Changes in steroid metabolite excretion via urine have been linked to starvation  and soditrations  or dehydAn important limitation of this study is that individuals were not treated with both the high and low dose of ACTH, which makes dose-dependent responses more difficult to interpret due to potential individual variability in stress perception. Having all individuals treated with the high and low dose would have been useful in better understanding dose-dependent responses in the species. Thus, a study with more individuals  will be useful to elucidate if the individual differences are due to dose, sex, or other factors which have not been considered.This study has identified suitable EIAs for the non-invasive measurement of stress-related hormone concentrations in naked mole-rats. Based on the results presented here, we recommend using fecal samples when quantifying GCMs in the species. Speculatively, the results here suggest that there are possible sex-related differences in GCM output in relation to dose-dependent ACTH treatment. The limitations of this study are the relatively low numbers of animals investigated. More research using a larger sample size is required to understand the underlying mechanisms which cause the differences in male and female responses to high and low doses of ACTH.


Polydimethylsiloxane (PDMS) has been widely used to make lab-on-a-chip devices, such as reactors and sensors, for biological research. Real-time nucleic acid testing is one of the main applications of PDMS microfluidic chips due to their high biocompatibility and transparency. However, the inherent hydrophobicity and excessive gas permeability of PDMS hinder its applications in many fields. This study developed a silicon-based polydimethylsiloxane-polyethylene-glycol (PDMS-PEG) copolymer microfluidic chip, the PDMS-PEG copolymer silicon chip (PPc-Si chip), for biomolecular diagnosis. By adjusting the modifier formula for PDMS, the hydrophilic switch occurred within 15 s after contact with water, resulting in only a 0.8% reduction in transmittance after modification. In addition, we evaluated the transmittance at a wide range of wavelengths from 200 nm to 1000 nm to provide a reference for its optical property study and application in optical-related devices. The improved hydrophilicity was achieved by introducing a large number of hydroxyl groups, which also resulted in excellent bonding strength of PPc-Si chips. The bonding condition was easy to achieve and time-saving. Real-time PCR tests were successfully conducted with higher efficiency and lower non-specific absorption. This chip has a high potential for a wide range of applications in point-of-care tests (POCT) and rapid disease diagnosis. Polymerase chain reaction (PCR) is a universal analysis methodology in molecular biology. It detects target amplicons by producing copies of specific deoxyribonucleic acid (DNA) molecules or fragments in vitro ,2. The fIn the 21st century, many pandemics, such as Ebola, coronavirus 2019 (COVID-19), and the monkeypox pandemic, have garnered worldwide attention to public health. Real-time PCR and real-time reverse transcription PCR  have returned to public sight and have become the standard molecular biological methods to diagnose these infectious diseases. The outbreak of COVID-19 has led to a research and industry boom in RT-PCR for detecting the associated virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,15. SimiMicrofluidics refers to a system that manipulates a very small volume of fluids on the microscale, which was gradually established after the Miniaturized Total Analysis System (μTAS) was first proposed by Manz ,20,21. OPDMS is one of the most common and cost-effective substrates with high clarity and a simple manufacturing process among all these materials. Due to its biocompatibility, high transparency, air permeability, adjustable rigidity, and relatively low cost, this material is suitable for a wide range of applications, particularly in micro-patterning . PatternHowever, the permeability and high hydrophobicity of the remaining PDMS surface still hinder the application of these chips. In molecule-related applications, such as biomolecular testing and drug screening, the inherently high hydrophobicity and gas permeability results in the non-specific absorption of molecules and proteins, leading to inaccurate results in these experiments . These p2 separation, and relative permittivity enhancement [A few studies have reported PDMS modification through the pre-mixing method to achieve desirable surface properties. One of the earliest reports by Xiao et al. added Poly (lactic acid)-poly(ethylene glycol) (PLA-PEG) as additives to increase the wettability of PDMS . Later, ancement ,55,56. Gancement . This smancement . AlthougThis study explores the application of PDMS-PEG copolymer to real-time PCR. We bonded the patterned material with Si to develop the PDMS-PEG copolymer silicon chip (PPc-Si chip) for real-time PCR diagnosis. PDMS-PEG copolymer was synthesized and made into patterned reaction chips for PCR by soft lithography. The patterned slides were activated by air plasma . Slides The Polydimethylsiloxane (PDMS) base and curing agent used in this study was Sylgard 184 silicone elastomer kit from Dow Corning. Dimethylsiloxane-(60–70% ethylene oxide) block copolymer was purchased from Gelest. SU-8 2050 photoresist was from Kayaku Advanced Materials Inc. Silicon wafers were provided and cut by the Nanosystem Fabrication Facility of the Hong Kong University of Science and Technology. ® base, hydrochloric acid , magnesium chloride (MgCl2), and Tween-20 surfactant, biological-grade water was purchased from Sigma-Aldrich. Deoxyribonucleotide triphosphates (dNTPs) in the 25 mM solution were ordered from ThermoFisher Scientific. The template we used is the Hepatitis B virus (HBV) plasmids synthesized by GenScript Biotech, which is a standard template for verifying PCR tests and material availability. Primers and TaqMan probes customized for the HBV plasmids were customized from GenScript Biotech following the sequence in 2 were dissolved into biological-grade water as 1 M and 0.05 M solutions, respectively. 1 M Tris-HCl buffer solutions at pH 8.09 were prepared with Trizma® base and hydrochloric acid. Primers and probes were mixed into a 100 µM solution (p-mix) for later use. We prepared the basic real-time PCR reaction system with a similar procedure and formula to our previous research . PotassiSU-8 photoresist was spin-coated on a 4-inch silicon wafer with a thickness of 250 µm SU-8 manufacturer’s instruction using spin coater . Symmetrical patterns of the reaction chip were fabricated on the wafer by photolithography  through the sequence of soft-baking, UV-irradiation, post-exposure baking, development, and hard-baking of the photoresist layer. PDMS base and curing agent were mixed on a ratio of 10:1, following adding a demanded volume of PDMS-PEG copolymer. PDMS-PEG copolymer concentrations of the pre-polymer mixture prepared in this study were 0, 0.25 wt%, 0.5 wt%, 0.75 wt%, 1 wt%, and 2 wt%. The mixture was poured on the SU-8-Si mold and degassed in a vacuum for 20 min, followed by the 24-h curing process at 80 °C to obtain patterned PDMS-PEG slides. The patterned slides were cut into 15 × 25 cm blocks. After treated patterns and silicon bases with air-plasma, as mentioned before, the treated surface of the two parts was brought to intimate contact and bonded together at 150 °C, and 0.25 MPa for 3 h. The resulting PPc-Si chips were ready to apply in real-time PCR tests. Another set of conventional PDMS-Si chips was manufactured following the same procedure that only excluded the addition of modifiers.The optical transparency of the modified patterned polymer with various content of PDMS-PEG was determined before the bonding process by ultraviolet-visible spectroscopy . We focused the incident light on the area of reaction chambers for each slide and recorded the transmittance in a wide wavelength from 1000 nm to 200 nm. In this study, we treated the transmittance data from 490 to 770 nm as references to choose the proper material for the real-PCR application.The area with and without pattern should have the same hydrophilicity. Therefore, the un-patterned part of the modified material was cut into 5 mm × 5 mm blocks for testing to ensure sufficient space for detection. Next, 4 µL water was dropped on the inner surface, and the contact angles were monitored for 10 min by a contact angle meter (Biolin Theta). The optical transmittance and CA results were considered as the basis for choosing the best additive content for the application in this study.The inner surface morphology of the PPc-Si chips was examined using a scanning electron microscope . Additionally, the surface roughness of the pattern surface to be bonded with silicon was observed by atomic force microscopy (AFM) imaging (Dimension Icon). An original PDMS slide with the same pattern was tested as a comparison.The elemental composition and chemical state of the PPc-Si reaction chambers were identified both before and after plasma treatment using X-ray photoelectron spectroscopy (XPS) with an Axis Ultra DLD X-ray photoelectron spectrometer (Kratos). PDMS slides with the same pattern were also tested under the same condition as a reference.The bonding strength for the ready-to-use PPc-Si chips was measured using an air compressor with an in-build barometer (Daertuo XDT550). A needle connected to the compressor was injected through the inlet of the reaction chamber while the chamber outlet was not open. The air pressure increased gradually at a rate of 5 psi/min. The pressure was recorded when the bonding interface cracked, or the material was broken. Both PPc-Si chips and conventional PDMS-Si chips bonded under the same condition were tested.4)2SO4, 0.04 μL of 0.05 M MgCl2, 0.16 μL of 25 mM dNTPs, 2 units of Taq polymerase, 0.04 μL of 100 mM p-mix, and 1 × 104 copies of corresponding templates were mixed. Each reaction unit was diluted with PCR-grade water to a final volume of 10 μL and injected into the PPc-Si chips. Chip inlets and outlets were sealed with another plasma-treated PDMS block. Simultaneously, the same reactions were processed in chips made of unmodified PDMS with the same pattern as a reference (Control A). The reactions in chips were performed in the SWM-02 real-time PCR system for PCR chips (Shineway). All reactions were conducted with 45 heating cycles, as we described before [In this study, we defined the required PCR reaction mix for one reaction in a chamber as 1 reaction unit. For each reaction unit, 0.5 μL 1 M KCl, 0.5 μL 1 M Tris−HCl (pH 8.09), 0.10 μL of 1 M  in situ for 10 min. After balancing surface hydrophilicity and transparency, we selected a BCP modifier concentration of 0.75 wt% to fabricate the PPc-Si chips and conducted the following characterization and practical tests.Surface morphology is crucial for a uniform bonding interface and firm bonding property. Weak surface uniformity can cause unbonded areas and affect bonding strength, increasing the risk of sample leakage during practical tests. We observed the surface morphology and height profile using SEM and SPM-AFM a,b. CompFor traditional PDMS blocks, plasma treatment activates silicon and polymer surfaces by increasing the hydroxy group content. Our modification introduced the PEG blockchain in the copolymer, resulting in higher hydroxy group content. The chemical states of patterned slides made with PDMS-PEG copolymer and original PDMS were characterized with XPS . From a During conventional real-time PCR tests, 40–45 repetitive heating cycles are processed, including two or three temperature stages from 55 °C to 95 °C for each cycle. This means that the inner pressure of the chip during the test will increase rapidly. We tested the pressure tolerance of the bonded PPc-Si chips and bonded chips with original PDMS patterned slides under the same bonding conditions. Each type of chip was tested four times, and the mean chip failure pressure was calculated. After bonding with the silicon wafer at 150 °C, 0.25 MPa for 3 h, the PPc-Si chips were all well bonded and could sustain a pressure of 4.52 (±0.01) bar, while the conventional PDMS-Si chips could tolerate 2.51 (±0.11) bar. Nevertheless, a 3% bonding failure rate was found for PDMS-Si chips. Furthermore, it should be emphasized that, for most of the PPc-Si chips, the chip failure was not caused by the splitting of the copolymer patterns and silicon wafers. Instead, the patterned copolymer slides themselves were crushed for many tested chips , meaningThe primary goal of this study was to determine the performance of PPc-Si chips in practical real-time PCR tests. Two sets of real-time PCR were conducted simultaneously on PPc-Si chips and conventional PDMS-Si chips to detect HBV plasmids. The same experiment was repeated several times, and nine curves from each set were selected and shown in The use of the PPc-Si chip eliminated the abnormal plateau stage, which corresponds to hydrophilicity, optimized gas permeability, and reduced molecule absorption. The average 0.5 cycle early in the Cq values also revealed the reduced biomolecular absorption of the PPc-Si chip compared to traditional PDMS-Si chips . The lowA silicon-based PDMS-PEG copolymer microfluidic chip, the PPc-Si chip, was developed for biomolecular diagnosis. PDMS-PEG copolymer was synthesized with varying content of the BCP modifier. Optical transmittance and contact angle of copolymers with various modifier content were observed. The transmittance decreased by about 4.25% at the entire tested wavelength range after adding 2% of the BCP modifier. In contrast, only about 0.8% of transmittance reduction occurred with a BCP modifier concentration of up to 0.75%. The wide-range evaluation of transmittance provided a reference for its optical property study and application in optical-related devices. The contact angle reduced to 90° within 45 s and rapidly dropped to 80° in 90 s when the modifier concentration was higher than 0.75%. The hydrophilicity switch started 15 s after contact with water at this concentration. The surface morphology of the patterned copolymer was similar to traditional PDMS patterns, with the roughness controlled within 20 nm. XPS results illustrated the high bonding strength with silicon wafers up to 4.52 bar. Real-time PCR tests for HBV were successfully conducted on PPc-Si chips with improved efficiency and optimized curve shapes, which benefited from the optimized surface properties and diminished non-specific absorption of molecules.Overall, the novel microfluidic chip developed in this study opens access to integrate silicon substrates with modified PDMS copolymers, extending the application of PDMS microfluidic devices that were previously hindered by high hydrophobicity and molecular absorption. The fabrication procedure is simple, cost-effective, and time-saving, and the bonding conditions for the chips are mild. This integrated chip shows high potential for a wide range of practical applications and significantly benefits point-of-care testing for food safety, environmental research, and disease diagnosis.


When viewing a completely ambiguous image, different interpretations can switch involuntarily due to internal top-down processing. In the case of the Necker cube, an entirely ambiguous stimulus, observers often display a bias in perceptual switching between two interpretations based on their perspectives: one with a from-above perspective (FA) and the other with a from-below perspective (FB). Typically, observers exhibit a priori top-down bias in favor of the FA interpretation, which may stem from a statistical tendency in everyday life where we more frequently observe objects from above. However, it remains unclear whether this perceptual bias persists when individuals voluntarily decide on the Necker cube's interpretation in goal-directed behavior, and the impact of ambiguity in this context is not well-understood. In our study, we instructed observers to voluntarily identify the orientation of a Necker cube while manipulating its ambiguity from low (LA) to high (HA). Our investigation aimed to test two hypotheses: (i) whether the perspective (FA or FB) would result in a bias in response time, and (ii) whether this bias would depend on the level of stimulus ambiguity. Additionally, we analyzed electroencephalogram (EEG) signals to identify potential biomarkers that could explain the observed perceptual bias. The behavioral results confirmed a perceptual bias in favor of the from-above perspective, as indicated by shorter response times. However, this bias diminished for stimuli with high ambiguity. For the LA stimuli, the occipital theta-band power consistently exceeded the frontal theta-band power throughout most of the decision-making time. In contrast, for the HA stimuli, the frontal theta-band power started to exceed the occipital theta-band power during the 0.3-s period preceding the decision. We propose that occipital theta-band power reflects evidence accumulation, while frontal theta-band power reflects its evaluation and decision-making processes. For the FB perspective, occipital theta-band power exhibited higher values and dominated over a longer duration, leading to an overall increase in response time. These results suggest that more information and more time are needed to encode stimuli with a FB perspective, as this template is less common for the observers compared to the template for a cube with a FA perspective. Processing visual information is an essential function of the brain that helps us interact with the environment. Neuroscientists agree that visual processing involves a combination of bottom-up and top-down components. The bottom-up component is driven by sensory processes that direct our attention to salient features of stimuli and process sensory information in visual areas. Invasive recordings have provided evidence that visual processing follows a bottom-up progression, from V1 to V4 , while the other has a “from-below” perspective (FB)  whether the perspective (FA vs. FB) influences response time, and (ii) whether the effect of perspective on response time depends on the level of stimulus ambiguity. Additionally, we conducted EEG analysis to identify biomarkers that can elucidate different processing mechanisms and explain perceptual bias. In summary, our behavioral results confirmed a shorter response time for the FA perspective and its disappearance under conditions of high ambiguity. The EEG analysis revealed differences in spectral power between the processing of Necker cubes with high ambiguity (HA) and low ambiguity (LA). Based on these observed differences, we proposed potential processing strategies for HA and LA stimuli and explained the response time bias in the goal-directed behavior.We conducted two experiments. Experiment 1 included a classical drawing of the Necker cube images SET1, . We measSixty-one healthy subjects  aged 18–33 years (20.2 ± 2.5 SD) with normal or corrected-to-normal visual acuity participated in the experiment. All of them provided written informed consent in advance. All participants were naïve. The study was approved by the local ethics committee of the Lobachevsky State University of Nizhny Novgorod  and was following the Declaration of Helsinki, except for registration in a database.bl = 1−a) and right-upper (br = a) squares on the 2D image, a∈ was a normalized edge's luminance in a gray-scale palette. Thus, the limit cases of a = 0 and a = 1 corresponded to unambiguous 2D projections of left- and right-oriented cubes, respectively, whereas a = 0.5 determined a completely ambiguous spatial orientation of the 3D cube. In our experiment, we used a set of the Necker cube images with a = {0.15, 0.25, 0.4, 0.45, 0.55, 0.6, 0.75, 0.85}  images a = {0.40, 0.45, 0.55, 0.60}, whose interpretations requires more effort. Unlike the other works on ambiguous stimuli processing , including all images . Then, we randomized indexes in this vector by using the function randperm in MATLAB. It returned a row vector containing a random permutation of the indexes from 1 to 400 without repeating elements. Finally, this randomized vector of indexes determined the order of stimuli presentation. We randomized the time of the stimuli presentations and pauses between them as tmin + rand*(tmax − tmin). Here, tmax and tmin defined minimal and maximal presentation/pause time, and rand is a MATLAB function that returns a single uniformly distributed random number in the interval . Each i-th stimulus exhibition lasted for a time interval of τ and varied from τmin = 1 s to τmax = 1.5 s. Pauses, γ between the subsequent presentations of the Necker cube images contained the abstract picture exhibition and varied from γmin = 3 s to γmax = 5 s  using a 48-channel NVX-52 amplifier . EEG signals were recorded from 32 standard Ag/AgCl electrodes , placed according to the international 10-10 system. The earlobe electrodes were used as a reference. The ground electrode was placed on the forehead. Impedance was kept below 10 KΩ. EEG was digitized with a sampling rate of 1,000 Hz. A band-pass FIR filter filtered the raw EEG signals with cut-off points at 1 Hz (HP) and 100 Hz (LP) and with a 50-Hz notch filter by embedded a hardware-software data acquisition complex. Eyes blinking and heartbeat artifacts were removed by Independent Component Analysis using EEGLAB software  aged 18–26 years  with no previous psychiatric or neurological history participated in the experiments. All subjects have normal or corrected-to-normal visual acuity. Similarly to Experiment 1, They provided written informed consent in advance. The study was approved by the local ethics committee of the Lobachevsky State University of Nizhny Novgorod and was following the Declaration of Helsinki, except for registration in a database.a = {0.15, 0.25, 0.4, 0.45, 0.55, 0.6, 0.75, 0.85}. All stimuli were mirrored around the horizontal axis. The task consisted of 16 stimuli : TOI1 was a 0.5 c interval following the stimulus presentation; TOI2 was a 0.5 s interval preceding the button pressing. The WP on these intervals was normalized using 1.5 s. prestimulus interval, so that we introduce event-related spectral perturbations (ERSP): ERSP = (WP − WPprestim)/WPprestim. All calculations were performed using the Fieldtrip toolbox in MATLAB . Thus, the response accuracy was above chance ensuring that participants based their decisions on the sensory information. For further confidence, we excluded all stimuli with erroneous responses. Similarly to Maksimenko et al. , we divipost-hoc analysis revealed that the subjects responded faster to the LA stimuli  than to HA ones : t(57) = 11.151, p < 0.001 (uncorrected) (t(57) = 2.962, p = 0.004 (uncorrected) (t(57) = 0.379, p = 0.706 (uncorrected) (t(57) = 4.246, p < 0.001 (uncorrected)  . They alrrected) . When thrrected) . In contrrected) .According to our previous work  and TOI2  in the following cases: (i) between HA and LA cubes; (ii) between the right- and left-oriented cubes; (iii) between the right- and left-oriented HA cubes; (iv) between the right- and left-oriented LA cubes.p < 0.001 in the frequency band of 1 − 3.5 Hz   . In the  TP9, P7 .p = 0.0126 in the frequency band of 3.75 − 5.75 Hz, including EEG sensors P8, O2  and reflected changes in the 3.75 − 4.5 Hz spectral power  in the frequency band of 8 − 13 Hz. The occipital theta reflected ERSP averaged across the occipital sensors  in the frequency band of 1 − 7 Hz. The frontal theta reflected ERSP averaged across the frontal sensors  in the frequency band of 1 − 7 Hz. We illustrated the time evolution of the ERSP in three spatial-frequency areas of interest during the intervals following the stimulus onset and preceding button pressing. The results are shown in Based on the results of statistical analysis, we defined three spatial-frequency areas of interest. The occipital theta shows an increase for both ambiguities but exhibits higher values for LA stimuli  was higher than the RT for the LA stimuli : t(17) = 6.952, p < 0.001 (uncorrected) (t(17) = 2.941, p = 0.01 (uncorrected) (t(17) = 0.142, p = 0.88 (uncorrected) (t(17) = 3.525, p = 0.003 (uncorrected)  . Studyinrrected) . Considedrawings , E. For drawings . For thedrawings .We conducted a perceptual decision-making task using ambiguous visual stimuli, specifically Necker cubes (Experiment 1). Participants were instructed to determine the orientation of the stimulus (left or right) and indicate their decision using their left or right hand. It is important to note that we excluded stimuli that were completely ambiguous from our study. As a result, the overall accuracy rate exceeded 90%, indicating a reliance on sensory information for decision-making and confirming the goal-directed nature of the task. The main finding of this experiment is that for stimuli with low ambiguity, participants responded faster to the left-oriented stimuli. However, for stimuli with high ambiguity, the response time remained relatively consistent between the left- and right-oriented stimuli.In the classical drawing of the Necker cube used in Experiment 1, the left-oriented stimulus has a from-above-perspective (FA), while the right-oriented stimulus has a from-below-perspective (FB). Therefore, both the orientation and perspective may contribute to the response time (RT) bias. To address this uncertainty, we conducted Experiment 2, where we presented both FA and FB projections for both left- and right-oriented stimuli. For the stimuli with low ambiguity, we found that participants had shorter response times for the FA perspective (both left- and right-oriented stimuli) compared to the FB perspective. Consistent with the findings of Experiment 1, this effect diminished for stimuli with high ambiguity. Together, our behavioral results demonstrate a perceptual bias toward the from-above (FA) perspective in goal-directed Necker cube viewing. This bias remains present for stimuli with low ambiguity but diminishes as ambiguity increases.Obtained behavioral results suggest that the brain utilizes different processing strategies for stimuli with low and high ambiguity. Specifically, in the case of low ambiguity, the processing strategy is more sensitive to the perspective compared to high ambiguity. To gain further insights into these processing strategies, we conducted a comparison of brain activity between low ambiguity (LA) and high ambiguity (HA) processing. Our findings reveal several important insights. First, processing the Necker cube, regardless of ambiguity level, leads to a reduction in alpha-band power across the occipital sensors . Additionally, ambiguous stimuli elicit higher ERD amplitudes compared to unambiguous stimuli . Second,The higher alpha-band ERD may reflect various processes, including alertness elicited by the stimulus and retrieval of information from memory for encoding  and involuntary processing  of ambiguous information. According to the predictive coding theory, the brain interprets information by continuously comparing sensory evidence with templates stored in working memory  have almost the same morphology. So, even if the Necker cube has an emotional impact on the observer, the emotional impact of different orientations barely differs, unlike the Rubin vase or face-non-face paradigm. Therefore, we expect that the state of the person will be the main source of emotional impact, rather than the emotional content of a stimulus.Our hypothesis is that the emotional state of an observer may change their problem-solving strategy for perceptual decisions when identifying interpretations of ambiguous stimuli. There is a view that people solve problems through either an analytical strategy or insight  perspective is more likely to be applied.In goal-directed processing, sensory information contains distinguishing features that differentiate between different interpretations. The observer actively controls information processing to ensure alignment between the presented information and the internal template. Participants successfully identify the Necker cube's interpretation for both from-above (FA) and from-below (FB) perspectives, indicating appropriate template selection based on sensory information. However, the increased response time observed for stimuli with the less common from-below (FB) perspective suggests the need for gathering additional information to achieve this alignment.Therefore, in goal-directed behavior, the longer response time observed for the less common from-below (FB) perspective compensates for the inherent top-down perceptual bias favoring the more frequent from-above (FA) perspective, which dominates during involuntary perception.The data that support the findings of this study are available from the corresponding author, VM, upon request.The studies involving humans were approved by Local Ethics Committee of the Lobachevsky State University of Nizhny Novgorod . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.VM and AH designed the study and wrote manuscript. AK analyzed data. SG and VK supervised experiments. AS, NG, AB, and VG conducted experiments. All authors contributed to the article and approved the submitted version.